Toluene (methylbenzene, C7H8) is a colorless aromatic solvent compound having a molecular weight of 92.14 Da, a melting point of −95° C., a boiling point of 110.8° C. and a specific gravity of 0.87 (15° C.), and giving off peculiar smell. Due to the superior solvent power and high evaporation rate, toluene is used in a variety of industrial fields such as formations of paints, printing ink, adhesives, and solvents for agrochemicals, and as a reactant material of various chemical reactions.
Upon exposure to toluene, individuals suffer from adverse effects such as a numbing of the olfactory sense, nausea, the irregular heart beat and the feeling of alcoholic intoxication. In severe cases, toluene exposure may cause hepatic and renal damage, neuropathy and cerebral injury. In recent years, the incidences of a rare occupational disease with symptoms of stiffening and sclerosing of all extremities and shoulders as well as the lungs were reported in workers who had been engaged in the adhesion work of tennis balls for about 10 years in a domestic tennis ball factory. It was found that the pathogenic cause of this disease was toluene intoxication due to the presence of toluene contained in adhesives.
At present, according to relevant provisions of the Industrial Safety and Health Act (ISH Act), the permissible exposure limit (PEL) of toluene is prescribed as TWA (Time-Weighted Average): 100 ppm. According to the Enforcement Decree of the Korea Foul Odor Prevention Act (effective from Feb. 20, 2005), the toluene emission limit will be restricted to within 30 ppm for industrial areas and within 10 ppm for other areas, from Feb. 20, 2008. Therefore, in order to comply with such environmental legislations for permissible discharge standards of toluene, factories and plants using toluene are forced to install local exhaust or process enclosure ventilation systems and to provide the respiratory protective devices in workplaces.
Meanwhile, in addition to such industrial problems associated with emission of toluene, inhalation of glue containing large amounts of toluene is a pathosociological phenomenon which is peculiarly observed in developing countries including Korea. Misuse of glue, which is committed particularly by young people, in order to experience phantasmagoria and hallucination similar to those appearing upon administration of narcotic drugs such as marijuana smoking and cocaine inhalation, is a serious social ill. The harmful evil influence of glue-sniffing on society and individuals may be more serious than that of the above-mentioned narcotic drug abuse, because glue-sniffing may cause incurable sequelae due to schizophrenia or brain atrophy caused by cerebral damage.
As discussed hereinabove, strictly strengthened regulations and practices in terms of industrial safety have led to an increasing demand for the development of a method which is capable of confirming exposure of workers to hazardous environmental factors under working conditions, such as toluene, and on the other hand, which is capable of determining whether young people and the like have inhaled toluene, in order to prevent and inhibit drug misuse and abuse.
Conventional methods currently used are based on determination of an amount of toluene per se or hippuric acid as a metabolite of toluene, which is present in the blood or urine, using analytical chemical methods. However, the performance of such determination methods have suffered from a need of expensive equipment such as the gas chromatography/mass spectroscopy (GC/MS) or the high performance liquid chromatography (HPLC) as well as highly skilled experts who can skillfully handle such equipments and can analyze the results thus obtained. Further, since application of such methods should involve long time-consuming processes such as preparation and concentration of samples through solvent extraction, it was hardly possible to apply such methods to large-sample groups such as worker groups, student groups or soldier groups.
In order to overcome the above-mentioned disadvantages and obtain objective examination results within a short period of time and with a simple method, use of immunoassays using antigen-antibody reactions is widely recommended. For detection of hippuric acid via the immunoassays, a specific antibody is necessary which is specifically reactive with hippuric acid present in the body fluid, particularly urine, but is not reactive with other components. However, when a molecular weight of an antigen is less than 3,000 Da, the activation of an immune system is not sufficiently achieved and therefore it is difficult to elicit the production of a specific antibody directed against the target antigen (Harlow and Land, 1988). In this respect, since hippuric acid has a low molecular weight of 179 Da and low immunogenicity, it is difficult to obtain the desired antibody with conventional immunization methods widely used in the art.
As already mentioned hereinbefore, the permissible exposure limit (PEL) of toluene in Korea is prescribed as 100 ppm (TWA) which corresponds to about 2.5 g/g of creatinine, as the content of hippuric acid in the urine is expressed in terms of creatinine in the urine. Meanwhile, according to clinical data from The Kyung-Hee University Medical Center (Seoul, Korea), the ordinary people excrete 1.6 L/day of urine in normal state. When it was calculated by taking into consideration 25% deviation between individuals, and by taking the above base value, i.e., 2.5 g/g of creatinine, as an average value, the urination amount is in the range of 1.2 to 2.0 L and therefore the amount of hippuric acid in the urine is about 1.25 to 2.08 mg/mL. As such, the hippuric acid-specific antibody intended for determination of this base value should not have a standard curve dynamic range in a low concentration (level of ng/mL or μg/mL) as typically shown in conventional antibodies, and therefore it is necessary to adjust the level of the antibody against hippuric acid to have the standard curve dynamic range (level of mg/mL) at which the presence/absence of toluene inhalation can be determined within the above ranges.
Upon absorption of toluene into the body, 20% of absorbed toluene is naturally eliminated via the respiratory system, whereas the remaining 80% of toluene is converted, via the microsomal mixed function oxidase system, into benzoyl alcohol which is, in turn, oxidized into benzoic acid via oxidative metabolism by alcohol dehydrogenase and aldehyde dehydrogenase. Finally, the resulting benzoic acid conjugates with glycine to form hippuric acid which is then excreted into the urine. In this connection, since the presence of benzoic acid in the body may also be due to food intake, a diagnostic strip according to the present invention should not exhibit detection sensitivity for trace amounts of hippuric acid which is excreted into the urine by means of benzoic acid absorbed into the body via daily dietary intake. Therefore, the hippuric acid-directed antibody is not simply intended to determine the presence of hippuric acid, and is thus preferably designed to be capable of determining whether or not hippuric acid is present over a predetermined level, depending upon various diagnostic purposes. That is, the antibody is preferable which can readily determine a sample having a diagnostic cut-off concentration (sensitivity level) of hippuric acid of more than about 1.25 mg/mL. Below 1.25 mg/mL of hippuric acid, there may be a problem of detecting excretion of hippuric acid due to other factors including dietary intake.
Until now, a great deal of attention has been focused only on the development of monoclonal antibodies (MAbs) which are capable of detecting a level of from ng/mL to μg/ml by a competitive assay. This is because insensitive antibodies having a low detection sensitivity of mg/mL level are highly cross-reactive with other antigens (proteins and the like) due to low specificity, and therefore are difficult to be used in diagnostic strips. As such, there is an urgent need for the development of an antibody having a low cross-reactivity, a specific reactivity only with hippuric acid and a titer capable of detecting hippuric acid at the level of mg/mL falling within the permissible exposure limit of toluene, with discrimination from hippuric acid remaining in the body via daily dietary intake.
Further, the existing methods for production of monoclonal antibodies are confined to animal experiments involving the use of experimental animals such as mice and the like, take a long period of time for the production of antibodies and require labor-intensive work for the selection and screening of large numbers of cells, as well as sufferings from difficulties in the control of specificity and affinity of the produced antibodies. To this end, there is a need for the development of a technique which is capable of securing genes for an antibody of interest using DNA recombinant techniques without animal experiments involving the use of experimental animals such as mice and the like, producing the desired monoclonal antibody in E. coli within a short period of time and engineering the specificity and affinity of the thus-produced antibody.